WO2022102787A1 - Procédé de production d'astrocytes à partir de cellules souches pluripotentes - Google Patents

Procédé de production d'astrocytes à partir de cellules souches pluripotentes Download PDF

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WO2022102787A1
WO2022102787A1 PCT/JP2021/042096 JP2021042096W WO2022102787A1 WO 2022102787 A1 WO2022102787 A1 WO 2022102787A1 JP 2021042096 W JP2021042096 W JP 2021042096W WO 2022102787 A1 WO2022102787 A1 WO 2022102787A1
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pluripotent stem
cells
neurog2
stem cells
gene
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治久 井上
孝之 近藤
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国立大学法人京都大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for producing astrocytes from pluripotent stem cells.
  • the present invention also relates to astrocytes obtained by this method.
  • the present invention relates to a cell preparation containing the above astrocytes.
  • Astrocytes are the cell type with the largest number of cells in the brain. Unlike nerve cells, it does not show electrical activity, so it has long been thought to be a "glue" -like entity that only fills the area around nerve cells. However, it is now clear that astrocytes are cells that play a variety of functions essential for maintaining homeostasis of the brain, such as regulation of synaptic transmission, immune response, and regulation of cerebral blood flow including the blood-brain barrier. ing.
  • Efforts have already been made to induce differentiation of astrocytes from pluripotent stem cells in order to elucidate the physiological functions of astrocytes and analyze the pathophysiology of related diseases. There are also reports from multiple groups on how to induce astrocytes differentiation from human iPS cells.
  • Non-Patent Document 1 a method of inducing astrocyte differentiation by culturing human pluripotent stem cells in the presence of a nerve differentiation-inducing factor and mimicking neurogenesis during the embryonic period was reported (Non-Patent Document 1). In this method, it takes several months (3 to 6 months) to obtain astrocytes because glial cells are generated after nerve cells are generated.
  • Non-Patent Document 2 discloses that astrocytes were obtained in 4 to 7 weeks by forcibly expressing NFIA or NFIA + SOX9 in human pluripotent stem cells using a CRISPER / Cas9 gene editing system.
  • Non-Patent Document 3 discloses that astrocytes were obtained in about 90 days by this method.
  • An object of the present invention is to provide a method for rapidly and highly efficiently producing astrocytes from pluripotent stem cells.
  • NEUROG2 gene Neurogenin 2 gene
  • the NEUROG2 gene is a gene that can induce differentiation of the cells into cerebral cortical neurons with almost 100% efficiency by forcibly expressing them in mouse pluripotent stem cells for 3 days or longer and in human pluripotent stem cells for 5 days or longer.
  • Patent Document 1 The present inventor has found that the nerve cell population obtained by this method contains a very small amount of cells having a glial-like morphology. Then, the present invention was completed by examining in detail the relationship between the expression period of the NEUROG2 gene and the appearance frequency of the glial-like cells.
  • the present invention includes the following.
  • [1] A method for producing astrocytes from pluripotent stem cells, wherein the following steps: (1) A step of expressing the NEUROG2 gene in pluripotent stem cells for less than 3 days. How to include.
  • [2] The method according to [1], wherein the expression period of the NEUROG2 gene is 8 hours or more.
  • [3] After the step (1), (2) A step of culturing the pluripotent stem cells without inducing the expression of the NEUROG2 gene.
  • the method according to [1] or [2] which comprises.
  • step (2) is a step of culturing in the presence of a factor that promotes WNT signaling.
  • the factor that promotes WNT signaling is one or more selected from the group consisting of BMP4 (bone morphogenetic protein 4), CNTF (Ciliary neurotrophic factor), and FBS (fetal bovine serum) [11].
  • BMP4 bone morphogenetic protein 4
  • CNTF Ciliary neurotrophic factor
  • FBS fetal bovine serum
  • the method described in. [13] The method according to any one of [1] to [12], wherein the pluripotent stem cell is a human induced pluripotent stem cell.
  • the human induced pluripotent stem cells are derived from a disease patient with an astrocyte abnormality.
  • the astrocyte produced by the method according to any one of [1] to [14].
  • astrocytes can be produced from pluripotent stem cells in a significantly shorter period than before and with high efficiency close to 100%.
  • the cells obtained by the method of the present invention can also be used as a cell preparation for the treatment of neurodegenerative diseases.
  • FIG. 1 shows a protocol for a method for producing astrocytes according to this embodiment.
  • the arrow indicates the timeline.
  • the medium conditions are shown above the timeline, and the Culture plate conditions (culture size and coating conditions) are shown below the timeline.
  • “NB” represents Neurobasal plus medium supplemented with B27 plus supplement
  • “AM” represents DMEM / F12 medium supplemented with N2 supplement, BMP4, CNTF, and FBS.
  • FIG. 2 shows immunostaining (green) of astrocyte marker GFAP on Day 12 and Day 18 against iAstrocytes produced from four types of NEUROG2-iPSC (HC1A, HC6B, N117E11, AD2S1) according to the method of FIG.
  • NEUROG2-iPSC H1A, HC6B, N117E11, AD2S1
  • FIG. 3 shows various astrocyte markers (GFAP, S100B, AQP4) and housekeeping genes (GAPDH,) for iAstrocytes produced from various types of NEUROG2-iPSC using the method of FIG. It is a graph which shows the result of having analyzed the transcription level of ACTB).
  • FIG. 3 represents the original iPS cell line name.
  • the leftmost shows the expression level in cells in which HC6B derived from a healthy person was cultured in a DOX-containing medium for 5 days to induce differentiation into nerve cells (iN).
  • FIG. 4 is a graph showing the results of analysis of the expression of nerve cell markers (TUBB3, MAP2, and SYN1) in the cells of FIG.
  • FIG. 5 is a diagram showing a protocol for an experiment in which NEUROG2-iPSC is cultured in a medium containing DOX for 2 days (Condition A), 3 days (Condition B), 4 days (Condition C), or 5 days (Condition D). .. “NB” and “AM” are synonymous with FIG. FIG.
  • FIG. 6 represents a typical image obtained by phase contrast microscopy for Day 8 cells obtained according to the protocol of FIG.
  • Conditions A to D show the same culture conditions as in FIG.
  • FIG. 7A represents a typical image obtained by immunostaining GFAP on iAstrocytes prepared from NEUROG2-HC1A according to the protocol of FIG.
  • FIG. 7B is a chart showing the results (calcium oscillation) of measuring the intracellular Ca ion concentration over time in the iAstrocyte shown in FIG. 7A.
  • the vertical axis is the signal intensity indicating the Ca ion concentration.
  • FIG. 8 is a chart showing the results of simultaneously recording the intracellular calcium ion concentration at 29 sites different from those in FIG.
  • FIG. 9 shows a co-immunostochemical image of GFAP immunostaining and DAPI staining for iAstrocytes obtained from iPS cells (A266) derived from Alexander disease patients and iPS cells (HC1A) derived from healthy subjects using the method shown in FIG. Represents A-E) and CD44 immunostaining image (F-K).
  • B is an enlarged image of the area surrounded by a square in A.
  • D and E are partially enlarged images of C.
  • FIG. 10 shows a Western blot showing the expression of GFAP protein in iAstrocyte.
  • hc1 and hc6 indicate astrocytes (Day 18) produced under the conditions of Example 1 from HC1A and HC6B, which are iPS cells derived from healthy subjects, respectively. Arrows indicate bands of GFAP protein.
  • FIG. 11 shows the effect of the expression period of the NEUROG2 gene on the induction efficiency of iAstrocyte.
  • hc1 and hc6 are the same as in FIG.
  • the error bar indicates the SD value.
  • the NEUROG2 gene means a gene generally known in the present art as the Neurogenin 2 gene.
  • the official genetic symbol is NEUROG2.
  • the structure of the nucleic acid encoding the NEUROG2 protein is exemplified in NCBI Accession Number: NM_024019 (human) or NM_009718 (mouse).
  • astrocytes can be defined as cells expressing one or more astrocyte marker genes, and examples of the marker genes include GFAP, S100B, Aquaporine4 (AQP4) and the like.
  • cells in which calcium oscillation is observed can also be referred to as functionally mature astrocytes.
  • Calcium oscillation in astrocytes is basically a spontaneous fluctuation of individual cells alone, but as it matures, it laterally adheres to surrounding astrocytes through intercellular adhesion or signal transduction by extracellular paracrine.
  • Directional oscillation propagation occurs. It is known that this lateral signal propagation (oscillation propagation) forms a network and plays an important role in the regulation of nerve cell activity and the control of vasoconstriction.
  • Producing astrocytes in the present invention means obtaining a cell population containing astrocytes, preferably containing 50%, 60%, 70%, 80%, 90%, or 95% or more of astrocytes. Is to obtain an astrocyte population.
  • the pluripotent stem cell is a stem cell having pluripotency capable of differentiating into a wide variety of cells existing in a living body and also having a proliferative ability, and is not particularly limited thereto.
  • embryonic stem (ES) cells embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem cells (GS cells), embryonic germ cells (EG cells), artificial pluripotent stems (iPS).
  • GS cells sperm stem cells
  • EG cells embryonic germ cells
  • iPS artificial pluripotent stems
  • Cells, cultured fibroblasts, pluripotent cells derived from bone marrow stem cells (Muse cells), etc. are included.
  • ES cells are pluripotent and self-replicating stem cells established from the inner cell mass of early mammalian embryos (eg, blastocysts) such as humans and mice.
  • ES cells are embryo-derived stem cells derived from the inner cell mass of the scutellum vesicle, which is the embryo after the morula at the 8-cell stage of the fertilized egg, and have the ability to differentiate into all the cells that make up the adult, so-called polymorphism. It has the ability and the ability to proliferate by self-replication.
  • Human ES cell lines for example, WA01 (H1) and WA09 (H9) are obtained from the WiCell Research Institute, and KhES-1, KhES-2 and KhES-3 are obtained from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan). It is possible.
  • sperm stem cells are testis-derived pluripotent stem cells, which are the origin cells for spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various lineages of cells, and have properties such as the ability to produce chimeric mice when transplanted into mouse blastocysts (M. Kanatsu-Shinohara et al. (M. Kanatsu-Shinohara et al.). 2003) Biol. Reprod., 69: 612-616; K. Shinohara et al. (2004), Cell, 119: 1001-1012).
  • Embryonic germ cells are cells with pluripotency similar to ES cells, which are established from primordial germ cells in the embryonic period.
  • Induced pluripotent stem (iPS) cells are made by introducing specific reprogramming factors into somatic cells in the form of DNA, RNA or protein, and are pluripotent and self-replicating proliferative bodies.
  • Cell-derived artificial stem cells K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al . (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International release WO 2007/069666).
  • Initialization factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1. , Beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 and the like can be exemplified.
  • iPS cells are used as pluripotent stem cells in the present invention
  • the embodiment in which human iPS cells are used is even more suitable.
  • Astrocytes produced from iPS cells derived from patients with diseases associated with astrocyte abnormalities are useful as model cells for the disease.
  • astrocytes produced by the method of the present invention can be used as an active ingredient of a cell preparation.
  • a cell preparation containing astrocytes produced from iPS cells of a healthy person or astrocytes produced from iPS cells modified to express a protein having a therapeutic effect is particularly suitable.
  • Such a cell product will be described in more detail in the section ⁇ Cell product> below.
  • the step (1) of the present invention is a step of directing the differentiation of pluripotent stem cells into astrocytes.
  • the NEUROG2 gene is expressed in pluripotent stem cells for a period shorter than the period required for the direction of differentiation into nerve cells, thereby directing the differentiation into astrocytes.
  • the period for expressing the NEUROG2 gene may be less than 3 days, preferably 8 hours or more and less than 3 days, more preferably 8 hours or more and 60 hours or less, still more preferably 24 hours or more and 60 hours or less, and particularly preferably 36 hours. Hours or more and 60 hours or less, most preferably 36 hours or more and 48 hours or less.
  • the NEUROG2 gene to be forcibly expressed may be either an endogenous NEUROG2 gene or an exogenous NEUROG2 gene, but an exogenous gene is preferable because of its ease of expression regulation.
  • a vector such as a virus containing a nucleic acid encoding NEUROG2, a plasmid, an artificial chromosome, or the like may be expressed by introducing it into pluripotent stem cells using a technique such as lipofection, liposome, or microinjection. Further, in step (1), cells into which the exogenous NEUROG2 gene has already been introduced may be used.
  • virus vector examples include a retro virus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, and a Sendai virus vector.
  • artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
  • plasmid vector a plasmid for mammalian cells can be used.
  • the above vector transposons before and after this expression cassette in order to excise the sequence encoding the NEUROG2 gene incorporated into the chromosome as needed. It may have a sequence.
  • the transposon sequence is not particularly limited, but piggyBac is exemplified as a suitable one.
  • These vectors can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the NEUROG2 gene can be expressed.
  • the pluripotent stem cell may contain a "nucleic acid encoding NEUROG2" functionally conjugated to an inducible promoter.
  • an inducible promoter include a drug-responsive promoter, and a suitable example thereof is the Tet-on promoter (tetO sequence having seven consecutive tetcycline responses), which is one of the tetracycline-responsive promoters. CMV minimal promoter with sequence (TRE)).
  • the promoter is a promoter that is activated by supplying tetracycline or a derivative thereof under the expression of a reverse tetracycline-regulated transactivator (rtTA: a fusion protein with reverse tetR (rTetR) and VP16AD). Therefore, when inducing the expression of the gene using a tetracycline-responsive promoter, it is preferable to use a vector having a mode of expressing the activator. Doxycycline (DOX) can be preferably used as the derivative of the tetracycline.
  • DOX Doxycycline
  • Expression-inducing systems using drug-responsive promoters other than the above include an expression-inducing system using an estrogen-responsive promoter (eg WO2006 / 129735) and RheoSwitch mammalian-inducible using a promoter induced by RSL1.
  • Expression system New England Biolabs
  • Q-mate system using a promoter induced by cumate
  • Cumate-induced expression system National Research Council (NRC)
  • ecdison responsive sequences examples thereof include a GenoStat-induced expression system (Upstate cell signaling solutions) using a promoter.
  • an expression vector having an expression-inducing system based on a drug-responsive promoter as shown above that is, a drug-responsive inducing vector
  • a drug capable of inducing activation of the promoter for example, the tetracycline responsiveness
  • the expression of NEUROG2 can be maintained by continuing to add tetracycline or DOX) to the medium for a desired period of time. Then, by removing the drug from the medium (for example, replacing it with a medium containing no such drug), it is possible to stop the expression of the gene.
  • NEUROG2 can be expressed in pluripotent stem cells by adding DOX to the medium.
  • the amount of DOX added here is not particularly limited, but is 0.01 to 100 ⁇ g / ml, preferably 0.1 to 10 ⁇ g / ml, and more preferably 1 to 5 ⁇ g / ml.
  • the basal medium or the basal medium to which the neurotrophic factor is added can be used.
  • Such basic media include Neurobasal Medium (Life Technologies), Glassgow's Minimal Essential Medium (GMEM) medium, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Medium (GMEM) medium.
  • GMEM Neurobasal Medium
  • GMEM Glassgow's Minimal Essential Medium
  • IMDM IMDM medium
  • Medium 199 medium Medium
  • EMEM Eagle's Minimum Essential Medium
  • ⁇ MEM medium Dulbecco's modified Medium
  • Medium Ham's F12 medium, RPMI1640 medium, Fischer's medium, and a mixed medium thereof are included.
  • the basal medium may contain serum or may be serum-free. If necessary, the medium may be, for example, B27 supplement (Invitrogen), B27Plus supplement (Invitrogen), Knockout Serum Replacement (KSR) (FBS serum substitute during ES cell culture), N2 supplement (Invitrogen), albumin, transferase. , Apotransferase, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, etc.
  • serum substitutes may contain one or more serum substitutes, lipids, amino acids, L-glutamine, Glutamax ( Invitrogen), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenic acid, progesterone and putresin, even if they contain one or more substances good.
  • Neurobasal Medium containing B27 supplement or a mixed medium of DMEM and F12 containing insulin, apotransferrin, selenic acid, progesterone and putrescine can be preferably used as the basic medium.
  • the culture temperature in the step (1) of the present invention is not particularly limited, but is about 30 to 40 ° C., preferably about 37 ° C., and the culture is carried out in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferable. Is about 2-5%.
  • the culture vessel is coated with extracellular matrix, as in the conventional method.
  • the extracellular matrix that can be used in the step (1) is not particularly limited as long as it is usually used for adhesive culture, and is, for example, poly-L-lysine, matrigel, vitronectin and a synthetic peptide derived from vitronectin, human type I. Examples thereof include collagen-like recombinant peptides, gelatin, laminin, collagen, and fibronectin.
  • the step (2) is a step in which the cells that have been directed to differentiate into astrocytes in the step (1) differentiate into astrocytes. Specifically, it is a step of culturing the cells without substantially expressing NEUROG2 (for example, without inducing the expression of NEUROG2).
  • the culture of step (2) may be carried out either in vivo or under conditions in the medium (in vitro). From the viewpoint of ease of controlling the differentiation rate, the in vitro culture step is preferable.
  • a medium containing a factor that promotes WNT signaling can be preferably used as the medium of the step (2).
  • Factors that promote WNT signaling promote differentiation into astrocytes, and the addition of such factors can increase the efficiency of astrocyte production.
  • Factors that promote WNT signaling include, but are limited to, BMP4 (bone morphogenetic protein 4), CNTF (Ciliary neurotrophic factor), and FBS (fetal bovine serum). It's not something.
  • the medium of step (2) may contain serum, and Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferase, It may contain one or more serum substitutes exemplified by apotransferase, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol and the like. In the present invention, it is a preferred embodiment to include a serum substitute, preferably a serum substitute for nerve cells such as N2 supplements.
  • KSR Knockout Serum Replacement
  • the medium of step (2) is Glutamax (Invitrogen), lipids, amino acids, L-glutamine, non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts. , Serenic acid, progesterone and putrecin and the like may also contain one or more substances.
  • subculture may be performed so as to have a low cell density.
  • An example of such low cell density may be a cell density of 2 ⁇ 10 4 to 10 ⁇ 10 4 cells / cm 2 per 48-well plate, preferably 5 ⁇ 10 4 cells / cm 2 cells per 48-well plate. Density. Culturing at low cell densities is preferred as it can reduce the likelihood that neurons will adhere to astrocytes and continue to survive (even under naturally non-adherable substrates).
  • the coating of the culture vessel may be gradually changed from one having a high affinity with cells to one having a low affinity with cells, and finally. May be uncoated.
  • PMSC PolyLlysine, Matrigel, Synthemax (Vitronectin-derived synthetic peptide, Corning), and Cellnest (human type I collagen-like recombinant peptide, Fujifilm)) coating (eg, Day1 to Day5), and then A gelatin-coated culture vessel (Day 6 to Day 12) may be used, and then an uncoated culture vessel may be used.
  • the uncoated culture vessel may be used after Day11, Day12, Day13, and Day14.
  • the culture period of step (2) is preferably 10 to 20 days, more preferably 10 to 16 days.
  • the culture temperature in the step (2) is not particularly limited, but is about 30 to 40 ° C, preferably about 37 ° C. Further, it is preferable that the culture is carried out in an atmosphere of CO 2 containing air, and the CO 2 concentration may be about 2 to 5%.
  • NEUROG2 is expressed in the step (1) for less than 3 days, preferably 2 days, and then administered in vivo without culturing in a medium (in vivo embodiment), astrocytes are generated in vivo. Is thought to occur. Therefore, such an in vivo aspect is also included in the present invention.
  • the present invention provides astrocytes produced by the method of the present invention.
  • the astrocyte may be referred to as induced Astrocyte (iAstrocyte) in order to distinguish it from naturally occurring astrocytes.
  • nerve cells that have been induced to differentiate from pluripotent stem cells may be referred to as induced neurons (iN).
  • the iAstrocyte according to the present invention is different from naturally-derived astrocytes in that a gene is introduced.
  • it is an iAstrocyte produced from human iPS cells.
  • the present invention provides a cell preparation containing astrocyte (iAstrocyte) produced by the method of the present invention as an active ingredient.
  • the cell preparation of the present invention containing iAstrocyte is effective for the treatment of neurodegenerative diseases, preferably neurodegenerative diseases associated with astrocyte abnormalities.
  • Neurodegenerative diseases associated with astrosite abnormalities include Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), spinal cerebral degeneration, polyline atrophy, and progressive hepatic palsy. Examples include primary degenerative types of nerve cells such as frontotemporal dementia, Huntington's disease, and spastic vs. paralysis, and primary degenerative types of astrosite such as Alexander's disease. Of these, it can be particularly suitably used for the treatment of Alexander disease, Alzheimer's disease and ALS.
  • Alexander disease is a rare hereditary neurodegenerative disease characterized by the presence of rosental fibers composed of glial fibrous acidic protein (GFAP), ⁇ B-crystallin, heat shock protein, etc. in astrocytes. Abnormal aggregates consisting of mutant GFAP or overexpressed GFAP are thought to be involved in Alexander's pathology. Clinically, it is classified into cerebral dominant type (type 1), medulla oblongata / spinal cord dominant type (type 2), and intermediate type (type 3) based on clinical symptoms and MRI imaging findings. Missense mutations in GFAP or deletions or insertions of several bases have been observed in 97% of Alexander's diseases, and genetic testing has been used as a definitive diagnostic method in recent years. Research is progressing on the elucidation of the pathophysiology of Alexander disease, but it is still insufficient.
  • GFAP glial fibrous acidic protein
  • ⁇ B-crystallin ⁇ B-crystallin
  • heat shock protein etc.
  • the cell preparation of the present invention can be used as a transplant therapeutic agent for treating the neurodegenerative disease.
  • iAstrocyte is produced as a parenteral preparation such as an injection, a suspension, and an infusion by mixing with a pharmaceutically acceptable carrier according to conventional means.
  • Pharmaceutically acceptable carriers that may be included in the parenteral preparation include, for example, isotonic solutions containing saline, glucose and other adjuvants (eg, D-sorbitol, D-mannitol, sodium chloride solution, etc.).
  • An aqueous solution for injection can be mentioned.
  • the preparation may be, for example, a buffer (eg, phosphate buffer, sodium acetate buffer), a soothing agent (eg, benzalconium chloride, prokine hydrochloride, etc.), a stabilizer (eg, human serum albumin, polyethylene glycol). Etc.), preservatives, antioxidants, etc. may be blended.
  • a buffer eg, phosphate buffer, sodium acetate buffer
  • a soothing agent eg, benzalconium chloride, prokine hydrochloride, etc.
  • a stabilizer eg, human serum albumin, polyethylene glycol. Etc.
  • preservatives, antioxidants, etc. may be blended.
  • iAstrocyte When formulating iAstrocyte as an aqueous suspension, add iAstrocyte to the above aqueous solution so that the content is 1 ⁇ 10 5 to 1 ⁇ 10 8 cells / ml, preferably 1 ⁇ 10 6 to 1 ⁇ 10 8
  • iAstrocyte transplantation can be performed by injecting the above suspension into lesions such as the cerebrum, medulla oblongata, and spinal cord.
  • the number of cells to be administered can be appropriately changed depending on the degree of lesion, etc., but for example, in the case of a patient with human Alexander disease, 1 ⁇ 10 5 to 1 ⁇ 10 8 cells, preferably 1 ⁇ 10 6 to 1 ⁇ 10 8 cells. Can be administered.
  • the cell preparation may contain cells immediately after undergoing the step (1) as an active ingredient.
  • the cells immediately after the step (1) can be differentiated into astrocytes in vivo and exert a therapeutic effect. Be expected.
  • the above transplantation treatment and drug therapy can be used in combination.
  • a concomitant drug for example, when the target disease is Alexander disease, it can be used in combination with an antiepileptic drug currently used as a symptomatic treatment for Alexander disease.
  • Such combination drugs can be used, for example, in the dosage and administration route usually used for the treatment of Alexander's disease.
  • NEUROG2-iPSC an iPS cell line in which a human NEUROG2 gene controlled by a Tet-on promoter is introduced into iPS cells established from a healthy subject or patient using piggyBac transposon.
  • NEUROG2-iPSC the exogenous NEUROG2 gene is inserted into the genome.
  • piggyBac transposon was used because the construct used in Patent Document 1 was available, and it has been confirmed that similar results can be obtained even with a transient expression system that is not inserted into the genome.
  • Table 1 shows the iPS cell lines used to prepare NEUROG2-iPSC and their origins.
  • NEUROG2-iPSC was cultured according to the conditions shown in FIG. 1 to produce astrocytes.
  • the start date of induction of differentiation into astrocytes that is, the start date of induction of expression of NEUROG2 may be set to Day 0, and the number of days thereafter may be represented by Day.
  • NEUROG2 The expression of NEUROG2 was induced by adding DOX to the medium at 2 ⁇ g / ml for 2 days from Day 0 to Day 2 unless otherwise specified.
  • NB medium Neurosporasal plus medium containing 0.5% B27 plus supplement
  • AM medium 1% N2 supplement, 10 ng / ml BMP4, 10 ng / ml CNTF, 2% FBS
  • DMEM / F12 medium containing was used.
  • Y-27632 was added for a predetermined period to 10 ⁇ M in order to reduce damage caused by medium replacement.
  • a PMSC-coated 48-well or 96-well plate was used for Day 0 to Day 5, and a gelatin-coated 6-well plate was used for Day 5 to Day 12.
  • NEUROG2-iPSC was seeded at 30 ⁇ 10 4 cells / cm 2 per 48-well plate, and the first passage was performed at the timing of changing the type of culture plate (Day 5). On Day 5, seeds were seeded at 5 ⁇ 10 4 cells / cm 2 per 48-well plate. After that, in addition to the timing of changing the type of the culture plate (Day 12), passage was appropriately performed according to the rate of cell proliferation.
  • Example 1 Production of astrocytes NEUROG2-iPSC produced from three iPS cell lines (HC1A, HC6B, N117E11) derived from healthy subjects and one iPS cell line (AD2S1) derived from familial Alzheimer's disease patients.
  • H1A, HC6B, N117E11 iPS cell lines derived from healthy subjects
  • AD2S1 iPS cell line derived from familial Alzheimer's disease patients.
  • GFAP immunostaining green
  • DAPI nuclear staining
  • the results are shown in FIG. With either NEUROG2-iPSC, only some cells were GFAP-positive on Day 12, but almost all cells were GFAP-positive on Day 18 (95% or more).
  • NEUROG2 when NEUROG2 was expressed in pluripotent stem cells for only 2 days, it rapidly differentiated into astrocytes from about 10 to 16 days after that. Furthermore, the GFAP-positive cells on Day 18 had a thicker cytoplasm and extended multiple protrusions than the GFAP-positive cells on Day 12, and were morphologically mature as astrocytes. Therefore, when NEUROG2 is expressed in pluripotent stem cells for a period shorter than the period required for nerve cell differentiation induction (for example, 2 days), it takes only 18 days from the start of NEUROG2 expression induction, which is much shorter than before. , Almost all cells have been shown to differentiate into morphologically mature astrocytes. In this example, the astrocytes obtained by forced expression of NEUROG2 may be referred to as "iAstrocyte" hereafter.
  • iAstrocytes were produced from NEUROG2-iPSC (Alex1-Alex3) derived from Alexander disease patients using the same method.
  • iN was also produced from HC6B NEUROG2-iPSC as a negative control.
  • the iN was produced by extending the period of culturing in a DOX-containing medium from 2 days to 5 days, and then culturing in a DOX-free NB medium (not AM medium).
  • the results of collecting each cell on Day 12 and analyzing the transcription levels of the astrosite marker genes (GFAP, S100B, and AQP4) and the housekeeping genes (GAPDH and ACTB) are shown in FIG.
  • the results of analyzing the transcription levels of MAP2 and SYN1) are shown in FIG.
  • the numerical value of the expression level on the vertical axis is a numerical value calibrated with the GAPDH expression level of FIG. 3 as 1.
  • NEUROG2 not only in pluripotent stem cells derived from healthy subjects but also in pluripotent stem cells derived from patients with neurodegenerative diseases caused by neurodegenerative and glia degeneration. It was shown that astrocytes can be induced to differentiate in days.
  • Example 2 Examination of NEUROG2 forced expression period (1)
  • NEUROG2-iPSC HC1A, HC6B
  • AD2S1, AD2EL NEUROG2-iPSC
  • DOX-containing medium 2, 3, 4, and 5 days.
  • the cells were cultured until Day 8 (Fig. 5, Condition AD).
  • a phase-contrast microscope observation was performed at the time of Day 8, and a typical phase-contrast microscope image obtained under each condition is shown in FIG.
  • Condition A cultured in Dox-containing medium for 2 days, iN (cells extending neurites) was hardly found.
  • iN was observed in Condition B with a culture period of 3 days in Dox-containing medium, the number of iN increased further in Condition C, and almost all cells extended long neurites in Condition D. It was iN.
  • the forced expression period of NEUROG2 suitable for inducing differentiation into astrocytes is less than 3 days, most preferably about 2 days.
  • Example 3 Examination of calcium oscillation
  • a phenomenon in which the Ca ion concentration in the astrocytes changes autonomously and regularly is observed, and neurotransmitter / regulatory factors such as glutamate, ATP, and serine are detected. It has been shown to regulate nearby astrocytes by releasing in a calcium concentration-dependent manner. Therefore, the presence or absence of calcium oscillation was analyzed as an index of the functional maturity of iAstrocyte.
  • iAstrocyte (Fig. 7A) obtained by culturing in DOX medium for 2 days, the Fluo-8AM body was incorporated into the cells and the intracellular Ca ion concentration was continuously recorded as the upper and lower fluorescence intensities and measured over time. .. The results are shown in FIG. 7B. As shown in FIG. 7B, it was confirmed that the Ca ion concentration changed up and down autonomously and regularly in the iAstrocyte, and calcium oscillation occurred.
  • FIG. 8 shows the results of recording calcium oscillations at multiple points at the same time.
  • the vertical axis of FIG. 8 represents the number assigned to each measurement site (ROI: Region of Interests). From this result, it can be seen that calcium oscillation occurs in various places in the iAstrocyte culture system. Furthermore, at the measurement sites 5, 6, 8, 9, 11-14, and 16 (particularly, 5, 6, and 12), the calcium concentration is high for about 190 seconds from the start of measurement. It was observed that the rising timings (vertical red dotted line in FIG. 8) tended to match.
  • astrocytes are known to form a network by propagating lateral calcium oscillations.
  • calcium oscillations were synchronized between multiple cells in iAstrocytes produced by this differentiation induction method. This result indicates that a communication network is formed between iAstrocytes.
  • the iAstrocyte produced by the method according to the present invention can spontaneously perform calcium oscillation and can also form an intercellular network via the oscillation. This indicates that the method according to the present invention can produce morphologically and functionally mature iAstrocytes.
  • Example 4 Production of cell model of neurodegenerative disease with astrocyte abnormality The presence or absence of GFAP-positive aggregates was examined for iAstrocytes produced from iPS cells (A266 strain) derived from Alexander disease patients. GFAP or CD44 was immunostained on Day 18 iAstrocytes produced from two types of NEUROG2-iPSC (A226, HC1A) by the method of Example 1. The results are shown in FIG.
  • GFAP-positive intracellular aggregates were observed in iAstrocytes produced from healthy subject-derived iPSC (HC1A) (Fig. 9A, B).
  • iAstrocytes produced from iPSC (A226) derived from Alexander disease patients GFAP-positive intracellular aggregates were frequently observed (Fig. 9C-E). That is, it was shown that astrocytes produced from iPSCs derived from Alexander disease patients by the method according to the present invention frequently spontaneously generate GFAP-positive intracellular aggregates characteristic of Alexander disease patients. Since the GFAP-positive intracellular aggregate is considered to contribute significantly to the onset of Alexander disease, it is considered that the astrocytes produced by the method according to the present invention can be a cell model of Alexander disease. ..
  • CD44 is a surface antigen marker expressed in glial progenitor cells and astrocytes. Whether it is an iAstrocyte (Fig. 9F-H) manufactured from a healthy person-derived iPSC (HC1A) or an iAstrocyte (Fig. 9I-K) manufactured from an Alexander disease patient-derived iPSC (A226), CD44 is present on the membrane surface. It was found to be distributed. This result suggests that astrocyte lineage differentiation is progressing in both iAstrocyte derived from healthy subjects and iAstrocyte derived from Alexander disease patients.
  • the production method according to the present invention can produce model cells for diseases associated with astrocyte abnormalities (for example, Alexander disease).
  • Example 5 Expression of GFAP protein in iAstrocyte It was confirmed by Western blot analysis that iAstrocyte expressed GFAP protein. Proteins were extracted (0.5 ⁇ g / ⁇ L) from cells on Day 18 of iAstrocytes produced under the conditions of Example 1 from healthy person-derived iPS cells HC1A and HC6B according to a conventional method, and a glass capillary electrophoresis device Wes (Protein simple) was used. Analyzed using. As the antibody, an antibody prepared using the full length of the human GFAP recombinant protein as an immunogen was used. The results are shown in FIG. It was confirmed that all iAstrocytes express GFAP.
  • Example 6 Examination of NEUROG2 forced expression period (2)
  • NEUROG2 forced expression period (2)
  • HC1A and HC6B which are iPS cells derived from healthy subjects, iAstrocytes were induced by shaking the culture period in a DOX-containing medium from 8 hours to 72 hours in the same manner as in Example 2.
  • the results are shown in FIG. It was clarified that astrocyte induction is possible even with expression at 8 hours.
  • the GFAP positive rate was substantially 0% when NEUROG2 was not forcibly expressed (data not shown).
  • morphologically and functionally mature astrocytes can be produced from pluripotent stem cells in a significantly shorter period of time than before and with high efficiency close to 100%.
  • the astrocytes produced by the method of the present invention are useful as model cells for analyzing the physiological function and role of astrocytes in pathological conditions, and as therapeutic agents (cell preparations) for diseases associated with astrocyte abnormalities.

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Abstract

La présente invention concerne un procédé de production d'astrocytes à partir de cellules souches pluripotentes, ledit procédé comprenant une étape d'expression du gène NEUROG2 dans les cellules souches pluripotentes pendant moins de trois jours.
PCT/JP2021/042096 2020-11-16 2021-11-16 Procédé de production d'astrocytes à partir de cellules souches pluripotentes WO2022102787A1 (fr)

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Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2014148646A1 (fr) * 2013-03-21 2014-09-25 国立大学法人京都大学 Cellule souche pluripotente pour l'induction de la différenciation neuronale
WO2019235576A1 (fr) * 2018-06-06 2019-12-12 富士フイルム株式会社 Procédé de production d'astrocytes a1 humains, astrocytes a1 humains et procédé d'évaluation d'une substance d'essai

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014148646A1 (fr) * 2013-03-21 2014-09-25 国立大学法人京都大学 Cellule souche pluripotente pour l'induction de la différenciation neuronale
WO2019235576A1 (fr) * 2018-06-06 2019-12-12 富士フイルム株式会社 Procédé de production d'astrocytes a1 humains, astrocytes a1 humains et procédé d'évaluation d'une substance d'essai

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Title
TCHIEU JASON; CALDER ELIZABETH L.; GUTTIKONDA SUDHA R.; GUTZWILLER EVELINE M.; AROMOLARAN KELLY A.; STEINBECK JULIUS A.; GOLDSTEIN: "NFIA is a gliogenic switch enabling rapid derivation of functional human astrocytes from pluripotent stem cells", NATURE BIOTECHNOLOGY, NATURE PUBLISHING GROUP US, NEW YORK, vol. 37, no. 3, 25 February 2019 (2019-02-25), New York, pages 267 - 275, XP036900609, ISSN: 1087-0156, DOI: 10.1038/s41587-019-0035-0 *
TETSURO YASUI, KINICHI NAKAJIMA: "Effect of oxygen concentration on acquisition of astrocyte differentiation ability of human pluripotent stem cell-derived neural stem / progenitor cells via epigenetics", FOLIA PHARMACOLOGICA JAPONICA, vol. 153, no. 2, 1 January 2019 (2019-01-01), JP , pages 54 - 60, XP009536668, ISSN: 1347-8397, DOI: 10.1254/fpj.153.54 *

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